Method for the intracellular delivery of biomolecules using thiocationic lipids

ABSTRACT

Lipid molecules bearing a cationic charge are described. These cationic lipids are useful in the delivery of biomolecules, such as oligonucleotides, nucleic acids, peptides, diagnostic imaging agents, proteins and drug molecules. In the form of liposomes, they can effectively be used for the intracellular delivery of biomolecules for therapeutic or diagnostic purposes.

FIELD OF THE INVENTION

The present invention relates to synthetic cationic lipophiliccompounds. In particular, it relates to thiocationic lipids that have apositively charged sulfonium ion, and the use of such thiocationiclipids as constituents of biomolecule conjugates and complexes, andpharmaceutical compositions thereof.

BACKGROUND OF THE INVENTION

Biomolecule-containing formulations, such as in vivo therapeutic agents,must be capable of exerting their effects on biological systems withoutcausing toxicity and without first being degraded by normal biologicalprocesses. This sometimes requires the use of complex delivery systemsand chemical modifications to make otherwise toxic and unstablecompounds more efficacious. In particular, pharmaceutical agents whichare highly charged, insoluble and/or high molecular weight sometimeshave limited pharmacological usefulness unless they are coupled to acarrier or incorporated into a delivery vehicle.

The problems associated with successful administration of pharmaceuticalformulations are compounded by the fact that certain types ofpharmaceutical agents cannot exhibit their biological effects unlessthey are taken up by cells. For example, the use of oligonucleotides asmodulators of gene function depends on their ability to interact withcellular components at the intracellular, and sometimes intranuclear,level. However, oligonucleotides, as well as other polyanionicsubstances, exhibit poor cellular uptake when delivered in aqueoussolution.

Many different approaches have been suggested for enhancingintracellular uptake of oligonucleotides. Some involve biologicalapproaches, such as the use of viral vectors (Cepko, et al., Cell 37:1053-1062 (1984)), or the covalent attachment of cell receptor ligands(Myers, et al., European Patent No. 273,085 (1988); Wu et al., J. Biol.Chem., 263: 14621-14624 (1988); and Vestweber, et al., Nature 338:170-172 (1989)). Others involve physical approaches, such as the directmicroinjection of DNA into cells (Capecchi, et al., Cell 22: 479-488(1980)) or cellular electroporation (Potter et al., Proc. Nat. Acad.Sci. 81: 7161-7165 (1984)). Yet others involve predominately chemicalapproaches, such as the attachment of lipophilic moieties (Shea, et al.,Nucleic Acid Research 18(13): 3777-3787 (1990); MacKellar, et al.,Nucleic Acid Research 20(13): 3411-3417 (1992)) or polypeptides(Stevenson, et al., J. Gen. Virol., 70: 2673-2682 (1989)).

Delivery vehicles such as liposomes have also been described for use inthe intracellular delivery of oligonucleotides. (Felgner, et al., U.S.Pat. No. 5,264,618 (1993); Eppstein, et al., U.S. Pat. No. 4,897,355(1990); and Wang, et al., Proc. Nat. Acad. Sci. 84: 7851-7855 (1987)).An advantage of using liposome formulations is the ability of thesesubstances to mimic naturally occurring cellular membrane substituents.This encourages fusion with cellular and endosomal membranes, whichresults in delivery of the liposome contents into the cytoplasm. Withoutsuch membrane fusion, extracellular substances are taken up byendocytosis and may be degraded within phagosomes before being releasedinto the cytoplasm.

Liposomes which are most useful for the intracellular delivery ofbiomolecules are often complex formulations containing mixtures ofdifferent lipophilic substituents. These complex mixtures allow foroptimization of the physical properties of the liposomes, such as pHsensitivity, temperature sensitivity and size. One recent advance is inthe recognition that certain pH sensitive amphiphilic compounds, such asdioleoylphosphatidyl-ethanolamine ("DOPE"), can be used to formulateliposomes which destabilize at acidic pH. This promotes fusion of theliposome with endosomal membranes when exposed to the degradative acidicpH and enzymatic contents of the endosome, which results in release ofthe contents of the lysosome into the cytoplasm. (See Ropert, et al.,Biochem. Biophys. Res. Comm. 183(2): 879-895 (1992); and Juliano, etal., Antisense Research and Development 2: 165-176 (1992)).

The inclusion of sterols in liposomes is also commonplace. In general,the presence of sterols in liposome formulations results in enhancedstability, both in vitro and in vivo. Liposome formulations for thedelivery of biomolecules which contain organic acid derivatives ofsterols, such as cholesterol or vitamin D₃, have been reported to beeasier to formulate than their non-derivatized water-insolubleequivalents (Janoff, et al. U.S. Pat. Nos. 4,721,612 and 4,891,208).However, in complex liposomal formulations containing certain polarlipids, the inclusion of such water soluble acid sterol derivatives maydestabilize liposomes and thus diminish efficacy.

Cationic lipids (i.e. derivatives of glycerolipids with a positivelycharged ammonium or sulfonium ion-containing headgroup) are particularlyuseful in liposomal formulations for the intracellular delivery ofnegatively charged biomolecules, such as oligonucleotides. Theirusefulness may be derived from the ability of their positively chargedheadgroups to interact with negatively charged cell surfaces. Thecationic lipid N- 1-(2,3-dioleyloxy)propyl!-N,N,N-trimethylammoniumchloride ("DOTMA") is described by Felgner et al. (Proc. Nat. Acad. Sci.84: 7413-7417 (1987); see also U.S. Pat. No. 4,897,355). Therein, acationic lipid having an ammonium group is used in a liposomeformulation to facilitate transfection of cells by polynucleotides. Inthese formulations, DOTMA has been shown to spontaneously interact withDNA to form ionic lipid-DNA complexes that are capable of fusing tonegatively charged lipids on cell surfaces, ultimately leading to theuptake of DNA by the cell.

Several other ammonium ion-containing cationic lipid formulations havealso been reported for such applications. These formulations include aDOTMA analog, 1,2-bis(oleoyloxy)-3-trimethylammonio)propane ("DOTAP")(Stamatatos et al., Biochemistry, 27: 3917-3925 (1988)); a lipophilicderivative of spermine (Behr et al., Proc. Nat. Acad. Sci., 86:6982-6986 (1989)); and cetyltrimethylammonium bromide (Pinnaduwage etal., Biochim. Biophys. Acta, 985: 33-37 (1989)) (See also Leventis etal., Biochim. Biophys. Acta, 1023: 124-132 (1990); Zhou et al., Biochim.Biophys. Acta, 1065: 8-14 (1991); Farhood et al., Biochim. Biophys.Acta, 1111: 239-246 (1992); and Gao, et al., Biochem. Biophys. Res.Commun., 179: 280-285 (1991)). Some commercially available cationiclipids include DOTMA (Gibco BRL, Bethesda, Maryland), DOTAP (BoehringerMannheim, Germany), and 1,2-diacyl-3-trimethylammonium propane ("TAP")(Avanti Polar Lipids, Birmingham, Alabama). However, many of theseammonium ion-containing lipids have been reported to be cytotoxic.

Sulfonium ions have entirely different physical properties than ammoniumions. In fact, ammonium ion-containing compounds are classified as hardbases, because the nitrogen atom possesses high electronegativity, ishard to polarize and oxidize, and the valence electrons are held tightlyby the nucleus. This characteristic may account for some of the toxicityassociated with ammonium ion-containing lipid formulations. In contrast,sulfonium ion- containing compounds are classified as soft bases,because the sulfur atom possesses low electronegativity, is easy topolarize and oxidize, and the valence electrons are held more loosely bythe nucleus. This decreased charge density exhibited by sulfoniumion-containing (i.e. "thiocationic") lipids may effectuate an enhancedinteraction with negatively charged cellular membranes, as well as adecreased toxicity.

It has also been suggested that one important feature which isassociated with the usefulness of lipids in enhancing transfection isthe size of their headgroups. Formulations containing cationic lipidswith "relatively small polar headgroups" have been predicted to be mostuseful (Felgner, et al., J. Biol. Chem. 269(4): 2550-2561 (1994)). Inaddition, Morris-Natschke, et al., (J. Med. Chem. 36: 2018-2025 (1993))describe the use of phosphocholine ether lipids, including a sulfoniumderivative, as anti-neoplastic agents, and report that the presence oflarge headgroup substituents decreased efficacy.

Because of the physiochemical properties of the sulfonium ion, athiocationic lipid with a larger headgroup may be preferred.Particularly, when the lipid headgroup consists of a sulfur atomsurrounded by adjoining saturated carbons, the sulfonium ion willexhibit a diffusion of charge to the neighboring carbons that mayfacilitate interaction of the lipid with cellular membranes, as well asdecreasing toxicity.

Although the previously described ammonium and sulfonium ion-containinglipids have been shown to be useful in many therapeutic applications,their incorporation into and use in the form of complex liposomalformulations has yet to be optimized. In particular, the enhancedefficacy which is achieved by incorporating Vitamin D into cationiclipid-containing formulations has not been explored.

It is therefore an object of the present invention to providethiocationic lipids which are less toxic in pharmaceutical formulationsthan their ammonium ion-containing counterparts.

It is also an object of the present invention to provide thiocationiclipid-containing pharmaceutical formulations which enhance theintracellular delivery of biomolecules to a greater extent thanpreviously described lipophilic compounds.

It is a further object of the present invention to provide cationiclipid-containing liposome formulations which demonstrate superiorefficacy.

SUMMARY OF THE INVENTION

The present invention relates to novel sulfonium ion-containing cationiclipids ("thiocationic lipids") and their use in pharmaceuticalformulations for the intracellular delivery of biomolecules. These novelcompounds are glycerolipids with headgroups which contain a sulfoniumion surrounded by at least three neighboring carbon atoms that serve tobeneficially diffuse the positive charge of the sulfonium ion.

In particular, the present invention relates to thiocationic lipids ofthe general formula: ##STR1## and optical isomers and/or salts thereofwherein: A¹ and A² are the same or different and are --O--CO--, --O--,--S--CO-- or --S--;

A³ is --O--, --O--CO--, --CO--O--, --S--, --S--CO--, --CO--S--,--O--CS--, --CS--O--, --CO--NH--, --NH--CO--, --CS--NH--, --NH--CS--,--NH--CO--O--, --NH--CO--NH--, --O--CO--NH--, or is absent;

R¹ and R² are the same or different and are H, or C₁ to C₂₃ saturated orpartially unsaturated alkyl or aralkyl, with the proviso that at leastone of R¹ and R² is not H;

R³ is a C₁₁, to C₁₂, alkyl, aralkyl, alkaryl, heterocyclyl orheteroaryl; or

R³ is an amino acid, a dipeptide, a tripeptide, a tetrapeptide or apentapeptide; or

R³ is ##STR2## wherein p is 1 to 5, q is 0 to 4, and R⁴ is H or a C₁ toC₄ alkyl; and

m, n and o are 0 to 8 with the provisos that m≧1 and m+n+o≧3.

These cationic lipids may be used in pharmaceutical formulations in theform of complexes with a biomolecule either alone or in combination withother lipid substituents. Alternatively, they may be covalentlyconjugated to biomolecules and used as such in pharmaceuticalformulations.

Pharmaceutical formulations according to the present invention consistof these complexes or conjugates and a pharmacologically acceptablecarrier. Examples of pharmacologically acceptable carriers includeaqueous solutions and complex delivery systems. Preferably, thepharmacologically acceptable carrier is a liposome.

An important aspect of the present invention is the discovery thatcertain cationic lipid-containing liposome formulations for use in theintracellular delivery of biomolecules exhibit enhanced efficacy. Suchliposomes generally consist of an ammonium or sulfonium ion-containinglipid, Vitamin D, a pH sensitive amphiphile, and a biomolecule.

Other features and advantages of the invention are apparent from thefollowing detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the viral inhibition of a liposome formulationcontaining DOMCATOP and an anti-HIV antisense oligonucleotide.

FIG. 2 illustrates the viral inhibition of a liposome formulationcontaining DODMECAP and an anti-HIV antisense oligonucleotide.

FIG. 3 illustrates the viral inhibition of a liposome formulationcontaining either cholesterol or Vitamin D3, and an anti-HIV antisenseoligonucleotide.

FIG. 4 illustrates the effects of liposome formulations containingeither DODMEHAP or DOMHYTOP and an anti-HIV antisense oligonucleotide oncellular metabolic activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a new class of synthetic thiocationiclipids that possess a sulfonium ion in the lipid head group. Thesethiocationic lipids are useful for enhancing the intracellular deliveryof biomolecules by serving as constituents of covalent conjugates and/orcomponents of pharmaceutical formulations. In order to more clearlydescribe the subject matter of the present invention, certain terms usedherein shall be defined as follows unless otherwise indicated:

Alkaryl: "Alkaryl" means an aryl group bearing at least one alkylsubstituent, for example tolyl or t-butylphenyl.

Alkenyl: "Alkenyl" means an alkyl group or moiety having at least twocarbons joined to each other by a double bond.

Alkyl: "Alkyl" means straight chain or branched chain hydrocarbon groupor moiety. When used alone, the term alkyl refers to a fully saturatedhydrocarbon group.

Alkynyl: "Alkynyl" means an alkyl group or moiety having at least twocarbons joined by a triple bond.

Amphiphilic: "Amphiphilic", when used to refer to an organic compound,means that the compound consists of both a hydrophobic (non-polar)moiety and a hydrophilic (polar) moiety. Examples of amphiphiliccompounds include sodium oleate; phosphatidylcholine and derivativesthereof, such as dioleylphosphatidylcholine ("DOPC"); anddioleylphosphatidylethanolamine ("DOPE").

Antisense Oligonucleotide: "Antisense oligonucleotide" means anoligonucleotide which is complementary to a target "sense" nucleic acid,and functions at least partially by sequence-specific mechanisms toregulate the functioning of the target nucleic acid.

Aralkyl: "Aralkyl" means an alkyl group to which is attached at leastone aryl ring moiety, for example benzyl, phenethyl or benzhydryl.

Aryl: "Aryl" means an aromatic hydrocarbon group or moiety, such asphenyl or naphthyl.

Biomolecule: "Biomolecule" means an organic compound which has adesirable biological activity or function, i.e. a "biological effect",in vivo. For example, biomolecules consisting of therapeutic agents mayalter cellular functions, such as gene functions. Alternatively,biomolecules consisting of diagnostic agents, such as magnetic resonanceimaging ("MRI") or computerized tomography ("CT") agents, have thebiological function of enhancing the diagnostic images of tissues and/ororgans.

Complementary: "Complementary", when used to refer to a nucleic acid,means a nucleic acid of one polarity containing a sequence ofnucleotides whose bases pair via Watson-Crick hydrogen bonds with thenucleotide bases of another nucleic acid of opposite polarity, i.e.adenine ("A") pairs with thymine ("T") or uracil ("U"), and guanine("G") pairs with cytosine ("C"). For example, a nucleic acid having thesequence GCAU in the 5' to 3' direction is "complementary" to a nucleicacid having the sequence CGTA in the 3' to 5' direction. Use of the termcomplementary herein is intended to include those nucleic acids whichare substantially complementary. Complementary nucleic acids can also bereferred to as one being the plus ("(+)") or "sense" strand and theother being the minus ("(-)") or "antisense" strand.

Complex: "Complex" means a non-covalent physical, usually ionic,association between two or more compounds. Examples of complexesinclude, for example, a negatively charged oligonucleotide which istonically associated with a cationic lipid.

DODMECAP: "DODMECAP" means1,2-dihexadecyloxy-3-(N-(5-carboxypentyl)-N,N-dimethylammonio)propane,and isomers and/or salts thereof.

DODMEHAP: "DODMEHAP" means1,2-dihexadecyloxy-3-(N-(6-hydroxyhexyl)-N,N-dimethylammonio)propane,and isomers and/or salts thereof.

DOMCATOP: "DOMCATOP" means S-((2,3-dihexadecyloxy)propyl)-S-(5-carboxypentyl)methylsulfonium, and isomers and/or salts thereof.

DOMHYTOP: "DOMHYTOP" means S-((2,3-dihexadecyloxy)propyl)-S-(6-hydroxyhexyl)methylsulfonium, and isomers and/or salts thereof.

DOPC: "DOPC" means dioleylphosphatidylcholine.

DOPE: "DOPE" means dioleylphosphatidylethanolamine.

Glycerolipid: "Glycerolipid" means a lipophilic molecule bearing aglycerol backbone, at least one hydrophobic tail, and which may alsocontain a hydrophylic polar headgroup.

Headgroup: "Headgroup" means that portion of a glycerolipid which isattached to the glycerol backbone at one of the terminal carbons.Headgroups can be neutral or polar.

Heteroaryl: "Heteroaryl" means an aromatic group bearing at least oneheteroatom as part of the aromatic ring structure, for example pyrroloor pyridyl.

Heterocyclyl: A cyclic group bearing at least one heteroatom as part ofthe ring structure, for example piperidinyl, pyrrolidinyl or morpholino.

Hybridize: "Hybridize" means the formation of a duplex betweencomplementary nucleic acids via base pair interactions.

Liposome: "Liposome" means a vesicle composed of amphiphilic lipidsarranged in a spherical bilayer or bilayers.

Modified: "Modified", when used to refer to a nucleic acid, means anucleic acid in which any of the natural structures have been altered.These include modifications to the phosphodiester linkages, the sugars(ribose in the case of RNA or deoxyribose in the case of DNA) and/or thepurine or pyrimidine bases. Modified phosphodiester linkages includephosphorothioates, phosphotriesters, methylphosphonates andphosphorodithioates.

Nucleic Acid Sequence: "Nucleic acid sequence", or "sequenc", means botha nucleic acid having a particular sequence of nucleotides, and also thesequence or order of nucleotides present in a particular nucleic acid.Which of these two meanings applies will be apparent form the context inwhich this term is used.

OBEHYTOP: "OBEHYTOP" means S-((2-benzyloxy-3-octadecyloxy)propyl)-S-(6-hydroxyhexyl)methylsulfonium, and isomers and/or saltsthereof.

OBECATOP: "OBECATOP" means S-((2-benzyloxy-3-octadecyloxy)propyl)-S-(5-carboxypentyl)methylsulfonium.

OA: "OA" means oleic acid.

Oligonucleotide: "Oligonucleotide" means a short segment of a nucleicacid.

Pharmacologically compatible carrier: "Pharmacologically compatiblecarrier" means a formulation to which a biomolecule can be added tofacilitate its administration to a patient without exhibiting anyunacceptable levels of toxicity or pharmacologically adverse effects.

Phosphorothioate oligonucleotide: "Phosphorothioate oligonucleotide"means an oligonucleotide having all phosphorothioate linkages in placeof naturally occurring phosphodiester linkages.

Polyanion: "Polyanion" means a molecule bearing more than one negativecharge.

Sequence: "Sequence" means the pattern or order of the nucleotide bases(A, G, C, T or U) in a nucleic acid.

Therapeutically effective amount: "Therapeutically effective amount"means an amount which is sufficient to demonstrate a biological effectwhich is sufficient to produce a desired therapeutic benefit.

The usefulness of the thiocationic lipids of the present invention isenhanced by the presence of a well-distributed positive charge in thelipid headgroup, which allows for efficient interaction with negativelycharged cellular membranes. The preferred use of these thiocationiclipids is in the intracellular delivery of negatively chargedbiomolecules. The thiocationic lipids also function to balance thenegative charges on such biomolecules which makes the resultantformulations more charge neutral. A particularly preferred use is in theintracellular delivery of oligonucleotides, for example phosphorothioateoligonucleotides. They may also be useful in the intracellular deliveryof certain diagnostic imaging agents, such as iodine-containing CTagents.

The thiocationic lipids of the present invention are designed to mimicnaturally occurring cellular membrane constituents. This allows thethiocationic lipid, and any associated biomolecule, to fuse with thecellular membrane thus facilitating intracellular delivery. Withoutbeing able to efficiently fuse with cellular membranes (which issometimes referred to as a substance's "fusogenic" property), foreignobjects are phagocytized and quickly degraded by lysosomal enzymes.

I. THIOCATIONIC GLYCEROLIPID STRUCTURE

The novel thiocationic lipids of the present invention can berepresented by the general formula of Structure I: ##STR3## and opticalisomers and/or salts thereof.

The thiocationic lipids given by Structure I can be described as havingthree separate moieties; a backbone, one or more tailgroups and aheadgroup. As given above, the backbone is composed of the three-carbonglycerol moiety; the tailgroups are given by R¹ and R², and are attachedto the backbone via A¹ and A² ; and the headgroup is given by; ##STR4##

The thiocationic glycerolipids of the present invention contain apositively charged sulfonium ion, S⁺. The sulfonium ion has theadvantage of providing a lower charge density as compared to apositively charged ammonium ion. In combination with the surroundingcarbons in the headgroup, the positive charge is diffused, thuspermitting enhanced interactions with negatively charged cell surfaces,as well as improved efficacy. Thus, m, n and o are each from 0 to 8,with the proviso that m≧1, and m+n+o≧3.

A¹ and A² provide the linkage between the glycerol backbone and thelipophilic tails. A¹ and A² can be the same or different and are--O--CO--, --O--, --S--CO-- or --S--. Particularly preferred embodimentsare compositions wherein both A¹ and A² are --O--, and R¹ and R² arelong chain (C16 to C18) alkyl moieties linked to A¹ and A² via a --CH₂--moiety. These long chain alkyl ether lipids are metabolically morestable than ester based lipids, and have been found to be superior intransporting nucleic acids into cells. A particularly preferred compoundof this type is DOMHYTOP, or its carboxyacid derivative, DOMCATOP. SeeExample I Parts A and B, respectively.

Another aspect of the present invention is the ability to vary thehydrocarbon chain length of the R² moiety in order to selectivelymanipulate the formation of preferred liposome structures. For example,when A² is --O-- and R² is H, the glycerolipid (i.e. "lysolipid") thusformed will have a single tailgroup. Use of lysolipids in combinationwith lipids having longer chain R² moieties may favor the formation ofliposomes having one or more bilayers. Use of lysolipids alone favorsthe formation of micelles. Such mixed lipid formulations can beoptimized for size and cellular uptake by varying the ratio ofcomponents accordingly.

Another preferred class of thiocationic lipids according to the presentinvention are compounds wherein A² is --O--, and R² is the aralkyl CH₂C₆ H₅. These benzyl derivatives have increased hydrophobicity due to thepresence of the aromatic moiety, and as such may exhibit preferredformulation characteristics. A particularly preferred compound of thisvariety is OBEHYTOP, or the equivalent carboxyacid derivative, OBECATOP.See Example I Parts C and D, respectively.

It is also possible to incorporate R¹ and R² moieties having varyingdegrees of unsaturation in order to optimize the fusogenic propertiesand formulation characteristics.

Other novel thiocationic lipids provided by the present invention aremolecules possessing additional cationic groups which are incorporatedinto the molecule in the R³ position. In the preferred embodiments ofthis family of cationic lipids, the additional cationic moieties arederived from the attachment of, for example, amino acids such as lysine,arginine, histidine or tryptophan.

The R³ is linked to the thiocationic lipid via A³, which may be --O--,--O--CO--, --CO--O--, --S--, --S--CO--, --CO--S--, --O--CS--, --CS--O--,--CO--NH--, --NH--CO--, --CS--NH--, --NH--CS--, --NH--CO--O--,--NH--CO--NH--, --CO--CO--NH--, or may be absent. The choice and/or needfor a linker would be easily determined by one of skill in the art.

The thiocationic lipids of the present invention can readily besynthesized using known methods. Typically, a lipid alcohol, such asthat derived from glycerol, is converted to its bromo derivative using abrominating reagent such as a mixture of carbon tetrabromide andtriphenyl phosphine. The bromo derivative thus formed is then reactedwith the sodium salt of an alkyl mercaptan, such as sodiumthiomethoxide, so as to obtain the alkyl thioglycerol intermediate.Alkylation of this intermediate is carried out via refluxing in thepresence of a haloalkyl compound using an appropriate solvent, ifnecessary, to provide the cationic lipid as its halide salt.

Thiocationic Lipid-Biomolecule Conjugates

The thiocationic lipids of the present invention can also be in the formof compositions comprising the thiocationic lipid covalently conjugatedto biomolecules.

The biomolecule can be any organic compound which exhibits a desirablebiological effect, and which is capable of retaining this biologicaleffect after conjugation to the cationic lipid. Examples of biomoleculesinclude, but are not limited to, proteins, hormones, genes,polypeptides, oligonucleotides, nucleosides, drugs, antibiotics,antibodies, diagnostic imaging agents, and derivatives and analoguesthereof. Covalent linkages between the thiocationic lipid and thebiomolecules can be accomplished using any known methods. See, forexample, PCT WO 94/05624.

The biomolecule is preferably an oligonucleotide, and more preferably aphosphorothioate oligonucleotide for purposes of resistance todegradative nucleases. In addition, the oligonucleotide may be RNA orDNA, but is more preferably DNA for purposes of resistance to RNases.Oligonucleotides which are useful in pharmaceutical formulations willtypically have a nucleotide sequence that is either of interest or issufficiently complimentary to hybridize to a nucleotide sequence ofinterest. The oligonucleotide typically is capable of performing abiochemical function in receptor host cells and/or altering theoperation of the cellular machinery. Examples of such functions are astransfection or antisense agents.

Although appropriate oligonucleotide length depends entirely on theparticular use for which the oligonucleotide is designed, certaingeneralizations are possible. If the oligonucleotide is to be used as anantisense therapeutic agent, it will preferably be between about 12 and50, and more preferably between about 15 and 30 nucleotides in length.

The oligonucleotides that may be used with the present inventioninclude, but are not limited to, naturally occurring nucleic acids, andmodified nucleic acids such as those having phosphorothioate,methylphosphonate or phosphorodithioate internucleotide linkages. Inaddition, the biomolecule can be a naturally occurring nucleoside, suchas adenosine, guanosine, cytidine, thymidine and uridine or analoguesthereof such as 5-fluorouridine, 5-alkyluridine, deazaguanosine,azaguanosine, azathymidine.

The site of attachment of a phosphorothioate oligonucleotide to thethiocationic lipids of the present invention may be via either the 5' or3' terminus, the internucleotide linkages, the nucleoside bases or thebackbone sugar moieties. Such linkages can be accomplished using anyknown methods. For example, see Goodchild, et al., BioconjugateChemistry 1(3) : 165-187 (1990). The cationic lipid may be attached viaA³ and/or R³ to the 3'-OH or 5'-OH. Alternatively, the cationic lipidmay be attached via A³ and/or R³ at any internucleotide linkage, such asvia the O or S atoms in the internucleotide phosphorothioate linkage. Inaddition, the cationic lipid may be attached to a base via A³ and/or R³to an endocyclic ring C or N, or to an exocyclic N or O. It may also beattached via A³ and/or R³ to the 1', 2' or 4' position of a sugarmoiety.

II. CATIONIC LIPID FORMULATIONS

The pharmaceutical formulations employing the thiocationic lipidsdescribed herein may be composed of thiocationic lipid-biomoleculeconjugates, or they may be composed of cationic lipid-biomoleculecomplexes which are formed by mixing together the thiocationic lipidsand the biomolecules described herein under conditions in which a stableassociation via ionic and/or hydrophobic interactions between the lipidand the biomolecule is formed. In both cases, the thiocationiclipid-biomolecule conjugates/complexes are prepared and administered ina pharmacologically acceptable carrier. Examples of pharmacologicallyacceptable carriers include aqueous solutions such as water, saline,buffers or carbohydrate solutions; and complex delivery systems such asliposomes, microspheres, or emulsions.

III. LIPOSOME FORMULATIONS

The thiocationic lipid-containing biomolecule formulations of thepresent invention preferably consist of liposomes. Any of theaforementioned biomolecules can be encapsulated into liposomes. Inaddition, numerous biomolecule-containing liposome formulations aredescribed in the literature, and serve as examples of additionalbiomolecules suitable for encapsulation using the liposome formulationsdescribed herein.

Lipid aggregates can take the form of completely closed structures madeup of a lipid bilayer containing an encapsulated aqueous volume (i.e.unilamellar liposomes), or they may contain more than one concentriclipid bilayer separated by an aqueous volume (i.e. multilamellarliposomes). Each lipid bilayer is composed of two lipid monolayers, eachof which has a hydrophobic (nonpolar) "tail" region and a hydrophilic(polar) "head" region. In the bilayer, the hydrophobic "tails" of thelipid monolayers orient toward the inside of the bilayer, while thehydrophilic "heads" orient toward the outside of the bilayer. It iswithin the aqueous phase that the biomolecule becomes entrapped in theliposome, unless the biomolecule is in the form of a lipid-biomoleculeconjugate, in which case the biomolecule may become embedded within thebilayer.

Liposomes may be made by a variety of techniques known in the art. (See,for example, Bangham et al., J. Mol. Biol., 13: 238-252(1965)). Thesemethods generally involve first dissolving and mixing the lipids in anorganic solvent, followed by evaporation. Then an appropriate amount ofthe aqueous phase is mixed with the lipid phase, and then allowed toincubate for a sufficient time for the liposomes to form. The aqueousphase will generally consist of the biomolecule in suspension with othersolutes, such as buffers or sugars.

In addition to the biomolecule to be encapsulated, the liposomes of thepresent invention are comprised of the thiocationic lipids describedherein alone, a mixture of thiocationic lipids described herein or thethiocationic lipids of the present invention combined with other knownlipids, such as an anionic lipid (for example, phosphatidylglycerol,phosphaticid acid or a similar anionic phospholipid analog), or aneutral lipid (for example, phosphatidylcholine orphosphatidylethanolamine). The present liposomal formulations mayfurther include a lysolipid, such as lysophosphatidylcholine,lysophosphatidylethanolamine, or a lyso form of a cationic lipidspecies.

The liposome can also include optional substituents, such as sterols,glycolipids, tissue or organ targeting substances such as antibodies orproteins, fatty acids, or any other natural or synthetic lipophilic oramphiphilic compounds.

Suitable sterols for inclusion into the liposomes include, but are notlimited to, cholesterol and Vitamin D, and are included in the liposomeformulations as stabilizers.

The molar ratio of thiocationic lipid to total lipid must be sufficientto result in the formation of a liposome with an overall positivecharge. This amount will depend on the charge and amount of theencapsulated biomolecule, as well as the charge and amount of otherconstituents of the liposome. Generally, the liposome will contain fromabout 9:1 to 1:9 molar ratio of cationic lipid to total lipid, andpreferably about 1:2 to 2:1. The molar ratio of total lipid tobiomolecule is from about 200:1-100:1, and is preferably about 160:1.

IV. LIPOSOME FORMULATIONS WITH ENHANCED EFFICACY

An important aspect of the present invention is the discovery that theefficacy of cationic lipid-biomolecule liposome formulations is enhancedby the presence of Vitamin D, and at least one pH sensitive amphiphiliclipid. Both ammonium ion and sulfonium ion-containing cationic lipidsare useful in such liposome formulations for the intracellular deliveryof biomolecules.

The cationic lipid may be any of the thiocationic lipids describedherein, or any other known ammonium or sulfonium ion-containing cationiclipids. As described above, the cationic lipid may be used alone or incombination with other cationic, neutral or anionic lipids.

The Vitamin D may be Vitamin D₃ (also called "cholecalciferol"), or anyanalogue or derivative of Vitamin D which does not significantlydiminish the overall efficacy-enhancing effects of Vitamin D, hereincollectively referred to as "Vitamin D". Preferably, underivatizedVitamin D₃ is employed.

Some amphiphiles have the ability to change their charge as a functionof pH and are therefore "pH sensitive". For example, liposomal vesiclescontaining pH sensitive amphiphiles such as oleic acid and/or DOPE canchange their charge as a function of pH, while DOPC containing vesiclesdo not change their charge in a pH dependent fashion. The ability of anamphiphile to change its charge as a function of pH depends on the otherconstituents of the liposome. In particular, the degree of alkyl chainsaturation may have an effect. In addition, there may be ion-pairinginteractions between the negative charges of the amphiphile, such as thenegatively charged phosphate in DOPE, and the positively chargedheadgroups in the cationic lipids. This would leave the positivelycharged amino group in the DOPE susceptible to a change in charge as afunction of pH. (See Felgner et al., J. Bio. Chem. 269:1-12 (1994)).

The pH sensitivity of the amphiphile serves to enhance the fusogenicityof the liposomal vesicles with endosomal membranes within the cell(Ropert et al., Biochem. Biophys. Res. Commun. 183(2): 879-85 (1992)).Suitable pH sensitive amphiphilic lipids include, but are not limited toDOPE and oleic acid. Preferably, the pH sensitive amphiphile is oleicacid, but any lipophilic amphiphiles which are susceptible to a changein charge as a function of pH at or near physiological pH (around 6 to7.5) after incorporation into the liposome are suitable for use.

A particularly preferred liposome formulation for the intracellulardelivery of oligonucleotides consists of cationic lipid:oleicacid:Vitamin D₃ in the molar ratio of 10:5:2. Other molar ratios can bedesigned to maximize the efficacy of liposomal formulations consistingof different constituents and/or biomolecules. Such molar ratios caneasily be determined using known techniques. See, for example, LiposomeTechnologies, CRC Press, publishers, Gregory Gregoriadis, ed. (1984).

V. DELIVERY OF PHARMACEUTICAL FORMULATIONS

The thiocationic lipids of the invention can be used in pharmaceuticalformulations to deliver biomolecules by various routes and to varioussites in the animal body to achieve a desired therapeutic effect. Thesepharmaceutical formulations may consist of thiocationiclipid-biomolecule complexes and/or thiocationic lipid-biomoleculeconjugates in any of the aforementioned pharmacologically acceptablecarriers.

Local or systemic delivery of the formulation can be achieved byadministration comprising application or insertion of the formulationinto body cavities, inhalation or insufflation of an aerosol, or byparenteral introduction, comprising intramuscular, intravenous,intradermal, peritoneal, subcutaneous and topical administration.

Orally administered formulations may be in the form of solids, liquids,emulsions, suspensions, or gels, or preferably in dosage unit form, forexample as tablets or capsules. Tablets may be compounded in combinationwith other ingredients customarily used, such as talc, vegetable oils,polyols, gums, gelatin, starch, and other carriers.

Parenteral formulations intended for injection, either subcutaneously,intramuscularly, or intravenously, can be prepared either as liquids orsolid forms for suspension in liquid prior to injection, or asemulsions. Such preparations are sterile, and liquids to be injectedintravenously should be isotonic. Suitable excipients are, for example,water, dextrose, saline, and glycerol.

The formulations may also be administered in aerosol form to cavities ofthe body such as the nose, throat, or bronchial passages.

The ratio of biomolecule to the cationic lipid and the other compoundingagents in these formulations will vary as the dosage form and amountrequires.

Effective dosages of the formulations described herein depend on thebiomolecule and its desired biological activity, as well as theparticular formulation kinetics, composition, physical properties,desired therapeutic effect, subject weight, etc. Dosage optimization caneasily be performed using known method.

VI. THERAPEUTIC USES

The preferred therapeutic use of the biomolecule formulations describedabove is in the intracellular delivery of a therapeutic agent comprisinga liposome-encapsulated antisense oligonucleotide. A particularlypreferred therapeutic agent formulation which is useful in inhibitingHIV consists of a thiocationic lipid-containing liposomes as deliveryvehicles of the phosphorothioate oligonucleotide given by SEQ. ID.NO. 1. This oligonucleotide is complementary to the MRNA sequence givenby SEQ. ID. NO. 2 encoding the HIV REV protein. (See Peterson, et al.,Published PCT Application No. WO 95/03407.)

EXPERIMENTAL PROCEDURES

The present invention can be better understood by way of the followingexamples that are representative of the preferred embodiments, but whichare not to be construed as limiting the scope of the invention. Allchemicals used herein were purchased from Aldrich Chemical Co.,Milwaukee, Wis., unless otherwise noted.

EXAMPLE I SYNTHESIS OF CATIONIC LIPIDS

PART A: SYNTHESIS OF THE BROMIDE SALT FORM OF DOMHYTOP

Step 1. Synthesis of 1,2-O-Dihexadecyl-3-bromo-1,2-propanediol

In a 250 ml round bottom flask equipped with a magnetic stir bar, 1.92 g(3.6 mmoles) of dihexadecylglycerol (Sigma Chemicals) was dissolved into120 ml of toluene. To this solution were added 3.54 g (10.7 mmoles) ofcarbon tetrabromide and 2.80 g (10.7 mmoles) of triphenylphosphine andthe reaction mixture was stirred overnight (18 hr) at room temperature.The yellow suspension was filtered and the filtrate concentrated on arotary evaporator to afford a white solid. This residue was dissolvedinto toluene, washed once with saturated sodium chloride, dried overanhydrous magnesium sulfate and concentrated under vacuum on a rotaryevaporator to afford 2.5 g of the crude product as a white powder. Thiscrude product was purified by flash column chromatography on a silicagel 60 (E. Merck, Germany) column by sequential elution with 100 ml eachof hexane, 1% ethyl acetate in hexane, 2% ethyl acetate in hexane andfinally with 3% ethyl acetate in hexane. Fractions (8 ml) were collectedand screened by thin-layer chromatography ("TLC") and those fractionsthat contained pure product (silica gel, 5% ethyl acetate in hexane,Rf=0.59) were pooled. The pooled fractions were concentrated undervacuum on a rotary evaporator to afford a quantitative yield of the1,2-O-dihexadecyl-3-bromo-1,2-propanediol as a white powder.

Step 2. Synthesis of 1.2-O-Dihexadecyl-3-methylthio-1.2-propanediolRacemic 1,2-O-dihexadecyl-3-bromo-1,2-propanediol (from Step 1), 2.0 g(3.3 mmoles), was dissolved into 100 ml of dry tetrahydrofuran in a 250ml round bottom flask equipped with a magnetic stir bar. To thissolution was added 2.33 g (33.2 mmoles) of sodium thiomethoxide powderand the reaction mixture was stirred overnight at room temperature. Thereaction was monitored by TLC (silica gel, 5% ethyl acetate in hexane)and an additional 490 mg (7 mmoles) of sodium thiomethoxide was added tothe reaction. After an additional 3 hr, the mixture was filtered and thefiltrate was washed with 50 ml tetrahydrofuran. The combined filtrateswere concentrated under vacuum using a rotary evaporator to afford ayellow residue. This residue was dissolved into 50 ml chloroform and theorganic solution was washed twice with 25 ml of a concentrated solutionof sodium bicarbonate. The organic phase was then dried over anhydrousmagnesium sulfate and concentrated on a rotary evaporator under vacuumto afford the crude product as a pale yellow oily residue. The crudeproduct was purified by column chromatography on a silica gel 60 (E.Merck, Germany) column using a step gradient of 0 to 10% ethyl acetatein hexane. The fractions collected were screened by TLC. Those fractionscontaining pure product were pooled and concentrated to afford1,2-O-dihexadecyl-3-methylthio-1,2-propanediol in 90% yield.

Step 3. Synthesis of 1,2-O-Dihexadecyl-3-(S-methyl)-(6-hydroxyhexyl)-sulfonium bromide Racemic1,2-O-dihexadecyl-3-methylthio-1,2-propanediol (from Step 2), 1.0 g(1.75 mmoles), was dissolved into 120 ml toluene in a 250 round bottomflask equipped with a magnetic stir bar and a reflux condenser. To thissolution was added 3.17 g (17.5 mmoles) of 6-bromohexanol and thesolution heated at 135° C. for 3 days. The brownish solution was thenallowed to cool to room temperature and concentrated on a rotaryevaporator to afford a brownish oily residue. This residue was dissolvedinto 50 ml chloroform and washed twice with 30 ml of a saturated sodiumchloride solution. The chloroform layer was then dried over magnesiumsulfate. Upon removal of the magnesium sulfate by filtration, thesolution was passed through a bed of silica to remove the extremelypolar impurities in the solution. This solution was next concentratedunder vacuum to afford the crude product as a waxy residue. The crudeproduct was purified by column chromatography using a silica gel 60 (E.Merck, Germany) column and eluted sequentially first with 100 ml hexane,followed by 100 ml of 10% ethyl acetate in hexane and finally with 30%ethyl acetate in hexane. Fractions containing the product as determinedby TLC were pooled and concentrated to afford the bromide salt ofDOMHYTOP as a low melting yellowish solid in 88% yield.

PART B: SYNTHESIS OF THE BROMIDE SALT OF DOMCATOP

Racemic 1,2-O-dihexadecyl-3-methylthio-1,2-propanediol (prepared as inPart A, Steps 1 and 2), 1.0 g (1.75 mmoles), was dissolved into 120 mltoluene in a 250 round bottom flask equipped with a magnetic stir barand a reflux condenser. To this solution was added 3.41 g (17.5 mmoles)of 6-bromohexanoic acid and the solution heated at 135° C. for 4 days.The brownish solution was then allowed to cool to room temperature andconcentrated on a rotary evaporator to afford a brownish oily residue.This residue was dissolved into 50 ml chloroform and passed through abed of silica to remove the extremely polar impurities in the solution.The resulting solution was next washed with 30 ml of a saturated sodiumchloride solution. The chloroform layer was then dried over anhydrousmagnesium sulfate. Upon removal of the magnesium sulfate by filtration,the solution was concentrated under vacuum to afford the crude productas a yellowish oily residue. This crude product was purified by columnchromatography using a silica gel 60 (E. Merck, Germany) column, elutingsequentially with 100 ml of hexane, followed by 10 ml of 10% ethylacetate in hexane and finally with 30% ethyl acetate in hexane.Fractions from the 30% ethyl acetate in hexane eluates contained theproduct as determined by TLC and were pooled and concentrated to affordthe bromide salt of DOMCATOP as a low melting yellowish solid in 42%yield.

PART C: SYNTHESIS OF THE BROMIDE SALT OF OBEHYTOP

Step 1. Synthesis of 1-O-Octadecyl-2-O-benzyl-3-bromo-1,2-propanediol

In a 250 ml round bottom flask equipped with a magnetic stir bar, 2.00 g(4.6 mmoles) of 1-O-octadecyl-2-O- benzyl-glycerol (Bachem, Switzerland)was dissolved into 120 ml of toluene. To this solution were added 4.58g. (13.8 mmoles) of carbon tetrabromide and 3.62 g. (13.8 mmoles) oftriphenylphosphine and the reaction mixture stirred for 4 hours at roomtemperature. The yellow suspension was filtered and the filtrateconcentrated on a rotary evaporator to afford a white solid. Thisresidue was dissolved into 50 ml chloroform, washed twice with 30 mlsaturated sodium bicarbonate, dried over anhydrous magnesium sulfate andconcentrated under vacuum on a rotary evaporator to afford 2.5 g of thecrude product as a white powder. This crude product was purified byflash column chromatography on a silica gel 60 (E. Merck, Germany)column by sequential elution with 100 ml each of hexane, 1% ethylacetate in hexane, 2% ethyl acetate in hexane and finally with 3% ethylacetate in hexane. Fractions collected were screened by TLC and thosefractions that contained pure product (silica gel, 5% ethyl acetate inhexane, Rf=0.56) were pooled. The pooled fractions were concentratedunder vacuum on a rotary evaporator to afford a quantitative yield ofthe 1-O-octadecyl-2-O-benzyl-3-bromo-1,2-propanediol as a white powder.

Step 2. Synthesis of1-O-Octadecyl-2-O-benzyl-3-methylthio-1,2-propanediol

Racemic 1-O-octadecyl-2-O-benzyl-3-bromo-1,2-propanediol, 1.99 g (4.0mmoles), was dissolved into 100 ml of dry tetrahydrofuran in a 250 mlround bottom flask equipped with a magnetic stir bar. To this solutionwas added 2.8 g (40.0 mmoles) of sodium thiomethoxide powder and thereaction mixture stirred for 4.5 hours at room temperature. The reactionwas monitored by TLC (silica gel, 5% ethyl acetate in hexane; Rf=0.51).The mixture was filtered and the filtrate was washed with 50 mltetrahydrofuran. The combined filtrates were concentrated under vacuumusing a rotary evaporator to afford a yellow residue. This residue wasdissolved into 50 ml chloroform and the organic solution washed twicewith 30 ml of a concentrated solution of sodium bicarbonate. The organicphase was then dried over anhydrous magnesium sulfate and concentratedon a rotary evaporator under vacuum to afford the crude product as apale yellow oily residue. This crude product was purified bychromatography on silica gel 60 (E. Merck, Germany) using a stepgradient of 0 to 10% ethyl acetate in hexane. The fractions collectedwere screened by TLC. Those fractions containing pure product (silicagel, 5% ethyl acetate in hexane, Rf=0.51) were pooled and concentratedto afford 1-O-octadecyl-2-O-benzyl-3-methylthio-1,2-propanediol in 90%yield.

Step 3. Synthesis of 1-O-Octadecyl-2-O-benzyl-3-(S-methyl-S-(6-hydroxyhexyl))-sulfonium bromide

Racemic 1-O-octadecyl-2-O-benzyl-3-methylthio-1,2-propanediol, 1.02 g(2.2 mmoles), was dissolved into 120 ml toluene in a 250 ml round bottomflask equipped with a magnetic stir bar and a reflux condenser. To thissolution was added 3.98 g. (22.0 mmoles) of 6-bromohexanol and thesolution heated at 135° C. for 3 days. The brownish solution was thenallowed to cool to room temperature and concentrated on a rotaryevaporator to afford a brownish oily residue. This residue was dissolvedinto 50 ml chloroform and passed through a bed of silica to remove theextremely polar impurities in the solution. The resulting solution wasnext washed twice with 30 ml of a saturated sodium bicarbonate solution,followed by extraction with 30 ml of a saturated sodium chloridesolution. The chloroform layer was then dried over anhydrous magnesiumsulfate. Upon removal of the magnesium sulfate by filtration, thesolution was concentrated under vacuum to afford the crude product as ayellowish oily residue. This crude product was purified by columnchromatography using silica gel 60 (E. Merck, Germany), elutingsequentially with 100 ml of hexane, followed by 100 ml of 10% ethylacetate in hexane and finally with 30% ethyl acetate in hexane.Fractions containing the product as determined by TLC (silica gel, 30%ethyl acetate in hexane, Rf=0.41) were pooled and concentrated to affordthe bromide salt of OBEHYTOP as a low melting yellowish solid in 85%yield.

PART D: EXAMPLE 4: SYNTHESIS OF THE BROMIDE SALT OF OBECATOP

Racemic 1-O-octadecyl-2-O-benzyl-3-methylthio-1,2-propanediol (preparedas in Part C, Steps 1 and 2), 1.0 g (1.75 mmoles), was dissolved into120 ml toluene in a 250 round bottom flask equipped with a magnetic stirbar and a reflux condenser. To this solution was added 4.30 g (22.0mmoles) of 6-bromohexanoic acid and the solution heated at 135° C. for 3days. The brownish solution was then allowed to cool down to roomtemperature and concentrated on a rotary evaporator to afford a brownishoily residue. This residue was dissolved into 50 ml chloroform andpassed through a bed of silica to remove the extremely polar impuritiesin the solution. The resulting solution was next washed with 30 ml of asaturated sodium chloride solution. The chloroform layer was then driedover anhydrous magnesium sulfate. Upon removal of the magnesium sulfateby filtration, the solution was concentrated under vacuum to afford thecrude product as a yellowish oily residue. This crude product waspurified by column chromatography using silica gel 60 (E. Merck,Germany), eluting sequentially with 100 ml of hexane, followed by 100 mlof 10% ethyl acetate in hexane and finally with 30% ethyl acetate inhexane. Fractions containing the product as determined by TLC (silicagel, 30% ethyl acetate in hexane, Rf=0.36) were pooled and concentratedto afford the bromide salt of OBECATOP as a low melting yellowish solidin 40% yield.

PART E: SYNTHESIS OF THE BROMIDE SALT OF DODMECAP

Step 1. Bromination of 1,2-dihexadecyloxy glycerol

1 g of 1,2-dihexadecyloxy glycerol was dissolved in 80 ml toluene in a100 ml round bottom flask. To this solution, 1.84 g carbon tetrabromideand 1.46 g triphenylphosphine were added. The reaction mixture wasstirred at room temperature for 24 hr, and the progress of the reactionmonitored by thin-layer-chromatography (5% ethyl acetate in hexane).

The resulting yellowish solid was filtered and the filtrate concentratedon a rotary evaporator and dissolved in acetone. This step was repeatedto isolate the product in the form of a white solid (yield 900 mg).

Step 2. Formation of 1.2-dihexadecyloxy-3-dimethylamino glycerol.

900 mg of 3-bromo-1,2-dihexadecyloxy glycerol was dissolved in 30 mlchloroform in a 100 ml round bottom flask. 720 mg dimethylamine wasadded to yield an approximately 10 fold molar excess, and the reactionmixture stirred at room-temperature overnight. The progress of thereaction was measured by thin-layer-chromatography (30% ethyl acetate inhexane).

The reaction mixture was transferred to a separating funnel and washedtwice with saturated aqueous sodium chloride. The organic layer wasdried on magnesium sulfate and then filtered. The filtrate wasconcentrated on a rotary evaporator to yield 630 mg product.

Step 3. Formation of the bromide salt of DODMECAP

270 mg of 1,2-dihexadecyloxy-3-dimethylamino glycerol from Step 2, 0.628g bromohexanoic acid and 0.271 g potassium carbonate were dissolved in80 ml toluene in a 100 ml round bottom flask. The reaction mixture washeated to 120° C. in an oil bath and the conditions maintained overnightalong with continuous stirring. The progress of the reaction wasmonitored using thin-layer-chromatography (30% ethyl acetate in hexane)and was shown to be complete in 20 hr.

The reaction mixture was then concentrated on a rotary evaporator. Thewhite residue was dissolved in 30 ml chloroform and washed thrice in aseparating funnel with 30 ml deionized water. The organic layer wasdried using magnesium sulfate and filtered. The filtrate wasconcentrated on a rotary evaporator and a white, waxy product obtainedafter precipitation with acetone. (yield 400 mg).

PART F: SYNTHESIS OF A BROMIDE SALT OF DODMEHAP

630 mg of 1,2-dihexadecyloxy-3-dimethylamino glycerol from Part E, Step2, and 1.83 g bromohexanol were dissolved in 60 ml toluene in a 100 mlround bottom flask. The reaction mixture was heated to 110° C. in an oilbath along with continuous stirring and the conditions maintained for 3days. The progress of the reaction was monitored usingthin-layer-chromatography (30% ethyl acetate in hexane) and was shown tobe complete after 3 days.

The reaction mixture was concentrated on a rotary evaporator. Theresidue was dissolved in 30 ml chloroform and extracted with 3 washes ofsaturated aqueous sodium bicarbonate in a separating funnel. The organiclayer was dried on magnesium sulfate and filtered. The filtrate wasfurther concentrated on a rotary evaporator and purified on a Silica Gel60 packed column (EM Sciences, Gibbstown, N.J.). The product was elutedusing a 30% solution of ethyl acetate in hexane. Starting with 100%hexane, the concentration of ethyl acetate was gradually increased ineach successive wash to 30%. (yield of product: 1.25 g).

EXAMPLE II SYNTHESIS OF A DOMCATOP-OLIGONUCLEOTIDE ("LIPONUCLEOTIDE")CONJUGATE

PART A: SYNTHESIS OF THE NHS ESTER OF THE BROMIDE SALT OF DOMCATOP

The bromide salt of DOMCATOP prepared in Example I Part B (130 mg, 0.17mmole), was dissolved in 20 ml dry tetrahydrofuran, and 65 mg (0.56mmole) of N-hydroxysuccinimide and 116 mg (0.56 mmole) ofdicyclohexylcarbodiimide were added to the solution. The solution wasstirred under nitrogen for 20 hours at room temperature. Thedicyclohexyl urea formed upon reaction was filtered off and the filtrateconcentrated under reduced pressure to afford a residue that wasdissolved into chloroform. This solution was extracted with saturatedsodium bicarbonate, dried over magnesium sulfate and concentrated toafford a quantitative yield of the NHS ester of DODMECAP.

PART B: SYNTHESIS OF A 5'-AMINO TERMINATED OLIGONUCLEOTIDE

Synthesis of a 5'-amino terminated oligonucleotide consisting of SEQ.ID. NO. 1 was carried out using known standard phosphoramidite chemistryprocedures and a commercially available automated synthesizer.Cyanoethyl phosphoramidites of bases adenosine, guanosine, cytosine andtyrosine and an amino-linker phosphoramidite allowed for the synthesisof the oligonucleotide via automated synthesis on a controlled poreglass solid support. The last step in the synthesis of the 5'-aminoterminated oligonucleotides was the use of an aminolinkerphosphoramidite (Applied Biosystems, Inc., Foster City, Calif.) so as toafford upon coupling the 5'-amino terminated oligonucleotide.Deprotection of nucleoside bases, the O-cyanoethyl group and cleavage ofthe support bound oligonucleotide were performed in one step bytreatment with ammonium hydroxide at 55° C. for at least 15 hr.Concentration of the resulting ammonical solution afforded the 5'-aminoterminated oligonucleotide, which was dissolved in water and stored forsubsequent use.

PART C: COUPLING OF THE NHS ESTER OF DOMCATOP TO THE 5'-AMINO TERMINATEDOLIGONUCLEOTIDE TO FORM A LIPONUCLEOTIDE

The phosphorothioate oligonucleotide prepared in

Part B (2.2 mg in 300 μl of water) was precipitated by addition of 33 μlof 3M sodium acetate and 1 mL ethanol at -20° C. for at least 1 hour.The precipitated oligonucleotide was separated by centrifugation at 4°C. for at least 30 minutes. The supernatant was removed, theoligonucleotide-containing pellet dried on a speed vacuum for 5 minutesand dissolved into 490 μl of 0.25M HEPES solution (pH 8.1). To thissolution 210 μl of pyridine and 280 μl of a 25 mM solution of thethiocationic NHS ester in pyridine were added. The resulting mixture wasvortexed and allowed to react at 55° C. for at least 18 hours. Thesolution was transferred into a 10 ml tube, treated with 280 μl of 3Msodium acetate, 700 μl of water and 7.9 ml of ethanol at -20° C. for atleast 2 hours. The resulting suspension was centrifuged at 17,000 rpm at4° C. for 1 hour and the supernatant removed. Theoligonucleotide-containing pellet obtained was dried and dissolved into1 ml. water. This crude product was purified by reversed phase HPLCusing a C8 radial pak column with a gradient of increasing methanolconcentration in 0.1M ammonium acetate (pH 7).

Fractions containing the liponucleotide were pooled, concentrated andthe residue was dissolved into 1-2 ml. water, treated with 0.25 ml 3Msodium acetate and 8 ml ethanol at -20° C. overnight, to afford uponcentrifugation a pellet of the liponucleotide.

EXAMPLE III SYNTHESIS OF LIPOSOME FORMULATIONS

The liposome preparations described below contained indicated amounts ofDOPE (Avanti Polar Lipids, Inc., Alabaster, Ala.); cholesterol (AvantiPolar Lipids, Inc., Alabaster, Ala.); Vitamin D₃ (Aldrich Chemical Co.,Milwaukee, Wis.); oleic acid ("OA"; NuChek Prep Inc., Elysian, Minn.);and DODMECAP, DOMCATOP, DOMHYTOP, DODMECAP, OBEHYTOP and OBECATOPprepared as described above. All ratios given are molar ratios. Unlessotherwise indicated, the molar ratio of cationic lipid:titratableamphiphile:sterol used in the preparation of liposomes herein was10:5:2. The term "total lipids" refers to the sum of all of thecomponents of the liposome given above. The phosphorothioateoligonucleotide referred to below is given by SEQ. ID. NO. 1.

A total of 80 μmoles of the total lipids in their desired ratios wasdissolved in chloroform, and then dried and rehydrated with an aqueoussolution containing 1 ml of a 0.5 mM solution of the phosphorothioateoligonucleotide in phosphate buffered saline, pH 7.4. The preparationswere then vortexed and allowed to come to equilibration by overnightshaking at 37° C. to allow the vesicles to form, and then freeze-thawed.Following this, the multilamellar liposomal vesicles thus formed wereextruded through polycarbonate filters of an appropriate pore size toobtain liposomes with a single bilayer. To remove any unencapsulatedmaterial, the liposomes were gel-filtered through a sizing gel column.The total amount of encapsulated oligonucleotide was determined using ahybridization protection assay. (See Arnold, et al., PCT WO 89/02476.)

EXAMPLE IV CELLULAR UPTAKE OF LIPOSOME FORMULATIONS

A total of 2×10⁶ cells (U937 cells; ATCC, Rockville, Md.) in 2 mlRPMI-1640 medium (BioWhittaker, Walkersville, Md.) with 10% fetal bovineserum (GibcoBRL, Grand Island, N.Y.) were plated in a 6-well tissueculture plate and incubated at 35° C. in 5% CO₂. A 100 nM solution ofliposome-encapsulated or free oligonucleotide in phosphate bufferedsaline (pH 7.4) was introduced to the cells. After 48 hr, the cells werelysed, and the amount of oligonucleotide was determined as described inExample III.

The results indicated that liposomes which were prepared as described inExample III which contained the cationic lipids DODMECAP, DODMEHAP,DOMCATOP, DOMHYTOP, OBEHYTOP or OBECATOP in combination with oleic acidand Vitamin D₃ demonstrated similar oligonucleotide uptake results.

EXAMPLE V VIRAL INHIBITION ASSAY

A total of 2.8X10⁶ cells (Jurkat cells; ATCC) in RPMI-1640 with 10%fetal calf serum were added to two 15 tubes in a total of 10 ml. Thecells were centrifuged, the supernatant decanted, and cells wereresuspended in 1 ml of the above medium. A 4.0 ml sample of retrovirus(HIV-1_(IIIB)) was added to one tube, and 4.0 ml of medium alone wasadded to the control. The cells were incubated for 2 hr at 37° C. in anatmosphere of 5% CO₂. Free virus was washed from the cells and the cellswere resuspended in 35 ml of medium.

The infected and control cells were placed in 125 μl aliquots into a 96well tissue culture plate. Varying concentrations of liposomes preparedaccording to Example III, or free oligonucleotides, were added to thewells and the plates were incubated for 6 days at 37° C. in anatmosphere of 5% CO₂. The p24 protein levels were determined by anantigen capture assay (Coulter Immunology, Hialeah, FL; U.S. Pat. No.4,886,742) using manufacturer's specifications and controls. The resultsare depicted in FIGS. 1, 2 and 3, with "--" representing the liposomewith the oligonucleotide, "-▪-" representing the control liposome withno oligonucleotide and "-▾-" representing the free oligonucleotide.

In FIG. 1, results are presented for liposomes prepared using thethiocationic lipid DOMCATOP with or without the oligonucleotide, and forthe oligonucleotide alone. As shown, the liposome containing theoligonucleotide effectively inhibited viral replication as demonstratedby a significant reduction in p24 levels.

In FIG. 2, the results are presented with the ammonium ion-containinglipid DODMECAP. As shown, no inhibition in viral replication wasobserved.

In FIG. 3, the results are presented for liposomes prepared using theammonium ion-containing lipid DODMEHAP with either Vitamin D₃ orcholesterol as the sterol. As shown, the Vitamin D₃ -containingliposomes were unexpectedly more effective at inhibiting viralreplication than their cholesterol-containing equivalents.

EXAMPLE VI CELLULAR METABOLIC ACTIVITY ASSAY

To determine the effects on cellular metabolic activity as a measure ofthe toxicity of the different liposome formulations, an XTT assay wasperformed on the infected cells from Example V as follows: A 1 mg/ml XTT(2,3-bis 2-methoxy-4-nitro-S-sulfophenyl!-2H-tetrazolium-S-carboxanilideinner salt; Sigma Chemical Co., St. Louis, Mo.) solution in cell mediumwas prepared. To this, 5 μl of 5 mM phenazine methosulfate (SigmaChemical Co.) per ml of XTT solution was added. A 50 μl aliquot of theresultant mixture was added to each well, and the plates were incubatedfor 4 hr at 37° C. in an atmosphere of 5% CO₂. Then, 15 μl of TritonX-100 (Sigma Chemical Co.) was added to each well, and the opticaldensity (O.D.) at 450-650 nm dual absorbancy was read. Corrected O.D.measurements were determined by subtracting the appropriate controllevels.

As depicted in FIG. 4, using increasing concentrations of liposomesprepared as described in Example III, liposomes with the ammoniumion-containing lipid (DODMEHAP) began to show signs of toxicity (asevidenced by a decrease in O.D.) at much lower concentrations than theliposomes with the equivalent sulfonium ion-containing lipid (DOMHYTOP).

Other aspects, uses and advantages of the present invention will beapparent to those skilled in the art upon review of the presentdisclosure. Those skilled in the art also will recognize that numerouschanges can be made to the structures and methods described hereinwithout departing from the spirit of the invention. The following claimstherefore set forth the scope of the present invention, which claims arenot to be limited by the specific embodiments described above in thespecification.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTTCGGGCCTGTCGGGTCCCCTCGGG26                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCCGAGGGGACCCGACAGGCCCGAAG26                                                  __________________________________________________________________________

We claim:
 1. A method for facilitating the delivery of a biomoleculeinto a cell, said method comprising the steps of:a) providing acomposition comprising a biomolecule complexed with or conjugated to athiocationic lipid of the general formula: ##STR5## and optical isomers,salts, or a combination of optical isomers and salts of said formula,wherein: A¹ and A² are the same or different and are --O--CO--, --O--,--S--CO-- or --S--; A³ is --O--, --O--CO--, --CO--O--, --S--, --S--CO--,--CO--S, --O--CS--, --CS--O--, --CO--NH--, --NH--CO--, --CS--NH--,--NH--CS--, --NH--CO--O--, --NH--CO--NH--, --O--CO--NH--, or is absent;R¹ and R² are the same or different and are H, or C₁ to C₂₃ saturated orpartially unsaturated alkyl or aralkyl, with the proviso that at leastone of R¹ and R² is not H; R³ is a C₁ to C₁₂ alkyl, aralkyl, alkaryl,heterocyclyl or heteroaryl; or R³ is an amino acid, a dipeptide, atripeptide, a tetrapeptide or a pentapeptide;or R³ is ##STR6## wherein pis an integer from 1 to 5, q is an integer from 0 to 4 and R⁴ is H or aC₁ to C₄ alkyl; and each of m, n and o is an integer from 0 to 8 withthe provisos that m≧1 and (m+n+o)≧3; and b) contacting said compositionwith a cell to facilitate delivery of said biomolecule within said cell.2. The method of claim 1, wherein said cell is an animal cell.
 3. Themethod of claim 2, wherein said animal cell is in culture.
 4. The methodof claim 1 or 3, wherein said composition further comprises a liposomecontaining said biomolecule completed with or conjugated to saidthiocarionic lipid.
 5. The method of claim 4, wherein said biomoleculeis an oligonucleotide.
 6. The method of claim 1 or 3, wherein saidbiomolecule is complexed with said thiocationic lipid.
 7. The method ofclaim 1 or 3, wherein said biomolecule is conjugated to saidthiocationic lipid.
 8. The method of claim 1 or 3, wherein saidbiomolecule is a polyanion.
 9. The method of claim 1 or 3, wherein saidbiomolecule is an oligonucleotide.
 10. The oligonucleotide of claim 9,wherein said oligonucleotide contains one or more modified sugars, oneor more modified intemucleoside linkages, or one or more modified sugarsand one or more modified intemucleoside linkages.
 11. Theoligonucleotide of claim 10, wherein said modified intemucleosidelinkages are selected from the group consisting of phosphorothioate,methylphosphonate, phosphotriester, phosphorodithioate andphosphoselenate linkages.
 12. The oligonucleotide of claim 11, whereinsaid modified intemucleoside linkages are phosphorothioate linkages. 13.The method of claim 9, wherein said oligonucleotide is aphosphorothioate oligonucleotide.
 14. The method of claim 1 or 3,wherein:A¹ and A² are each independently selected from the groupconsisting of --O--, --O--CO--, and --S--; R¹ and R² are eachindependently selected from the group consisting of:i) a saturated orpartially unsaturated C₁ -C₁₂ alkyl group, ii) benzyl, and iii)phenethyl; A³ is selected from the group consisting of --O--, --CO--O--,--S--, and --NH--CO--, or is absent; R³ is selected from the groupconsisting of:i) saturated or partially unsaturated C₂ -C₂₃ alkyl oraralkyl, ii) -- (CH₂)_(p) --NR⁴ !_(q) --R⁴, wherein p is from 3 to 4, qis from 0 to 4 and R⁴ is H, iii) --(CH₂)_(p) --NR⁴ ₃ ⁺, wherein p isfrom 2 to 3 and R⁴ is methyl, or iv) ##STR7## wherein R₄ is methyl. 15.The method of claim 14, wherein:R¹ and R² are each independentlyselected from the group consisting of:i) a saturated or partiallyunsaturated C₂ -C₂₃ alkyl group, and ii) benzyl; A³ is --O--; and R³ is-- (CH₂)_(p) --NR⁴ !_(q) --R⁴.
 16. The method of claim 15, wherein:R¹and R² are each independently selected from the group consisting of:i) asaturated or partially unsaturated C₁₈ alkyl group, ii) a saturated orpartially unsaturated C₁₆ alkyl group, and iii) benzyl; andm=6.
 17. Themethod of claim 16, wherein:A¹ and A² are both --O--; R¹ is a saturatedC₁₆ or C₁₈ alkyl group; R² is a benzyl group; n=0; o=0; q=0; and R⁴ isH.
 18. The method of claim 14, wherein:R¹ and R² are each independentlyselected from the group consisting of:i) a saturated or partiallyunsaturated C₂ -C₂₃ alkyl group, and ii) benzyl; A³ is --CO--O--; and R³is -- (CH₂)_(p) --NR⁴ !_(q) --R⁴.
 19. The method of claim 18, wherein:R¹and R² are each independently selected from the group consisting of:i) asaturated or partially unsaturated C₁₈ alkyl group, ii) a saturated orpartially unsaturated C₁₆ alkyl group, and iii) benzyl; andm=5.
 20. Themethod of claim 19, wherein:A¹ and A² are both --O--; R¹ is a saturatedC₁₆ or C₁₈ alkyl group; R² is a benzyl group; n=0; o=0; q=0; and R⁴ isH.
 21. A composition for facilitating the delivery of a biomolecule intoa cell, said composition comprising a biomolecule complexed with orconjugated to a thiocationic lipid of the general formula: ##STR8## andoptical isomers, salts, or a combination of optical isomers and salts ofsaid formula, wherein;A¹ and A² are the same or different and are--O--CO--, --O--, --S--CO-- or --S--; A³ is --O--, --O--CO--, --CO--O--,--S--, --S--CO--, --CO--S--, --O--CS--, --CS--O--, --CO--NH--,--NH--CO--, --CS--NH--, --NH--CS--, --NH--CO--O--, --NH--CO--NH--,--O--CO--NH--, or is absent; R¹ and R² are the same or different and areH, or C₁ to C₂₃ saturated or partially unsaturated alkyl or aralkyl,with the proviso that at least one of R¹ and R² is not H; R³ is a C₁ toC₁₂ alkyl, aralkyl, alkaryl, heterocyclyl or heteroaryl; or R³ is anamino acids a dipeptide, a tripeptide, a tetrapeptide or apentapeptide;or R³ is ##STR9## wherein p is an integer from 1 to 5, q isan integer from 0 to 4, and R⁴ is H or a C₁ to C₄ alkyl; and each of m,n and o is an integer from 0 to 8 with the provisos that m≧1 and(m+n+o)≧3.
 22. The composition of claim 21, wherein said biomolecule isa polyanion.
 23. The composition of claim 21, wherein said biomoleculeis an oligonucleotide.
 24. The composition of claim 21 or 23, whereinsaid composition further comprises a liposome containing saidbiomolecule complexed with or conjugated to said thiocationic lipid. 25.The method of claim 21 to 23, wherein said biomolecule is complexed withsaid thiocationic lipid.
 26. The method of claim 21 or 23, wherein saidbiomolecule is conjugated to said thiocationic lipid.
 27. Theoligonucleotide of claim 23, wherein said oligonucleotide contains oneor more modified sugars, one or more modified internucleoside linkages,or one or more modified sugars and one or more modified internucleosidelinkages.
 28. The oligonucleotide of claim 27, wherein said modifiedinternucleoside linkages are selected from the group consisting ofphosphorothioate, methylphosphonate, phosphotriester, phosphorodithioateand phosphoselenate linkages.
 29. The oligonucleotide of claim 28,wherein said modified internucleoside linkages are phosphorothioatelinkages.
 30. The composition of claim 23, wherein said oligonucleotideis a phosphorotioate oligonucleotide.
 31. The composition of claim 21 or23, wherein:A¹ and A² are each independently selected from the groupconsisting of --O--, --O--CO--, and --S--; R¹ and R² are eachindependently selected from the group consisting of:i) a saturated orpartially unsaturated C₂ -C₂₃ alkyl group, ii) benzyl, and iii)phenethyl; A³ is selected from the group consisting of --O--, --CO--O--,--S--, and --NH--CO--, or is absent; R³ is selected from the groupconsisting of:i) saturated or partially unsaturated C₁ -C₁₂ alkyl oraralkyl, ii) -- (CH₂)_(p) --NR⁴ !_(q) --R⁴, wherein p is from 3 to 4, qis from 0 to 4 and R⁴ is H, iii) --(CH₂)_(p) --NR⁴ ₃ ⁺, wherein p isfrom 2 to 3 and R⁴ is methyl, or iv) ##STR10## wherein R⁴ is methyl. 32.The composition of claim 31, wherein:R¹ and R² are each independentlyselected from the group consisting of:i) a saturated or partiallyunsaturated C₂ -C₂₃ alkyl group, and ii) benzyl; A³ is --O--; and R³ is-- (CH₂)_(p) --NR⁴ !_(q) --R⁴.
 33. The composition of claim 32,wherein:R¹ and R² are each independently selected from the groupconsisting of:i) a saturated or partially unsaturated C₁₈ alkyl group,ii) a saturated or partially unsaturated C₁₆ alkyl group, and iii)benzyl; andm=6.
 34. The composition of claim 33, wherein:A¹ and A² areboth --O--; R¹ is a saturated C₁₆ or C₁₈ alkyl group; R² is a benzylgroup; n=0; o=0; q=0; and R⁴ is H.
 35. The composition of claim 31,wherein:R¹ and R² are each independently selected from the groupconsisting of:i) a saturated or partially unsaturated C₂ -C₂₃ alkylgroup, and ii) benzyl; A³ is --CO--O--; and R³ is -- (CH₂)_(p) --NR⁴!_(q) --R⁴.
 36. The composition of claim 35, wherein:R¹ and R² are eachindependently selected from the group consisting of:i) a saturated orpartially unsaturated C₁₈ alkyl group, ii) a saturated or partiallyunsaturated C₁₆ alkyl group, and iii) benzyl; andm=5.
 37. Thecomposition of claim 36, wherein:A¹ and A² are both --O--; R¹ is asaturated C₁₆ or C₁₈ alkyl group; R² is a benzyl group; n=0; o=0; q=0;and R⁴ is H.